PROJECT SUMMARY Translation of messenger RNAs (mRNAs) into proteins by the ribosome and the rest of the translation machinery (TM) is a fundamental step in gene expression that is central to life. Because the bacterial TM is a proven target for the development of new antibiotics and because many human diseases have been causally linked to dysregulation of translation, the mechanisms of translation and translational control in bacteria and eukaryotes remain under intense investigation. Over the past two decades, structural studies have revealed the large-scale structural rearrangements the TM undergoes during protein synthesis. Unfortunately, the size, complexity, and conformational flexibility of the TM have greatly impeded studies of these dynamics, significantly limiting our understanding of how these dynamics contribute to the mechanisms of translation and translational control. Nonetheless, using a combination of single-moleucle fluorescence and structural techniques, we and others have been able to characterize the dynamics of the core steps of translation by the bacterial TM. Despite these accomplishments, critical gaps in our understanding remain regarding whether and how the dynamics of these core steps are modulated as part of biomedically important translational control strategies. To fill these gaps, in the first aim of this application, we propose to use a combination of single- molecule fluorescence, structural, and biochemical approaches to investigate how the dynamics of the bacterial TM are modulated in order to drive ribosomal frameshifting. Frameshifting is a translational control strategy in which the TM slips backward or forward by one or more nucleotides on the mRNA to either correct an insertion or deletion ?frameshift? mutation that would otherwise result in production of an aberrant or truncated protein or to drive the synthesis of more than one protein product from a single mRNA. These experiments promise to reveal the still-elusive mechanism(s) that underlie frameshifting. In the second aim, we will use analogous approaches to investigate how ribosome rescue factors modulate the dynamics of the bacterial TM as part of the mechanisms through which they recognize and rescue ribosomes that have become translationally compromised. These studies will provide structure-based mechanistic models of bacterial ribosome rescue systems that can be exploited in the development of new antibiotics. In the third aim, we will extend our combination of single-molecule fluorescence and structural techniques to a yeast translation system, enabling us to investigate eukaryotic-specific aspects of the core steps of translation, frameshifting, and ribosome rescue. The results of these studies will reveal the mechanisms that drive and regulate translation in eukaryotes, providing a framework for investigating the role of translational control in human health and disease.